Microarray-based rapid cloning of an ion accumulation deletion mutant in Arabidopsis thaliana
Microarray-based rapid cloning of an ion accumulation deletion mutant in Arabidopsis thaliana
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DOI：
10.1073/pnas.0404780101
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发表时间：
2004-10-26
影响因子：
11.1
通讯作者：
Schroeder, JI
中科院分区：
文献类型：
--
作者：
Gong, JM;Waner, DA;Schroeder, JI
在这里，我们描述了一种基于微阵列的定位策略的发展，以快速分离缺失突变基因。这种方法对于定位难以表型的突变基因特别有用。这一策略使用掩蔽批量分离分析来掩盖不相关的缺失，从而允许通过微阵列杂交来自WT和突变F-2群体的汇集基因组DNA来识别目标缺失。元素图谱已被证明是分离拟南芥中营养和有毒金属积累突变体的有力工具。通过基因芯片定位，发现一个钠过量积累突变体FN1148在AtHKT1基因的第二外显子和内含子中存在523个碱基的基因组缺失。进一步的分离、互补和不同盐敏感突变体之间的比较分析证实，AtHKT1基因的缺失是导致FN1148突变体地上部钠过量积累和叶片钠敏感性的原因。这些结果表明，基于微阵列的克隆是快速克隆离子积累或其他难以表型定位的基因缺失突变体的有效工具，如代谢突变体或细胞信号突变体。
Here we describe the development of a microarray-based mapping strategy to rapidly isolate deletion mutant genes. The presented approach is particularly useful for mapping mutant genes that are difficult to phenotype. This strategy uses masking bulk segregant analysis to mask unrelated deletions, thus allowing identification of target deletions by microarray hybridization of pooled genomic DNA from both WT and mutant F-2 populations. Elemental profiling has proven to be a powerful tool for isolation of nutrient and toxic metal accumulation mutants in Arabidopsis. Using microarray mapping, a sodium overaccumulation mutant FN1148 was identified as having a 523-bp genomic deletion within the second exon and intron of the AtHKT1 gene. Further cosegregation, complementation, and comparative analyses among different salt-sensitive mutants confirmed that the deletion within the AtHKT1 gene is responsible for the sodium overaccumulation in shoots and leaf sodium sensitivity of the FN1148 mutant. These results demonstrate that microarray-based cloning is an efficient and powerful tool to rapidly clone ion accumulation or other genetic deletion mutants that are otherwise difficult to phenotype for mapping, such as metabolic or cell signaling mutants.